To observe the effect of high glucose stimulation on the expression of guanylate exchange factor NHX1 in mouse kidney cells, and to explore the role of NHX1 in high glucose-induced podocyte injury and its possible molecular mechanism. The expression of NHX1 in podocytes of diabetic nephropathy patients was observed by immunofluorescence staining and laser confocal microscopy. The immortalized podocytes were cultured in vitro, and the podocytes were stimulated with high glucose for 48 h. RT-PCR, Western blot and immunization were used. Fluorescence detection of mRNA and protein expression of NHX1 in podocytes stimulated by high glucose; Western blot, immunofluorescence and scratch assay were used to detect the expression of NHX1 and the expression of podocin, the activity of podocytes and the nuclear access of TRPV5. The transcription of the target gene downstream of TRPV5 was detected by RT-PCR. NHX1 expression was significantly decreased in podocytes and high glucose-stimulated podocytes of diabetic nephropathy patients (P<0.05). After silencing NHX1, podocyte marker protein podocin expression was significantly decreased and podocyte activity was increased. The nuclear translocation of TRPV5 increased, and the transcription of the target gene downstream of TRPV5 increased (P < 0.05). In contrast, the expression of overexpressed NHX1 group was significantly increased, the activity of podocytes was decreased, the nuclear access of TRPV5 was decreased, and the transcription of the target gene downstream of TRPV5 was decreased (P<0.05). NHX1 may reduce podocyte injury in diabetic nephropathy by inhibiting TRPV5 entry into the nucleus.